Peptide, Complex of Peptide and siRNA, and Methods of Use Thereof

ABSTRACT

A series of peptides and a peptide-siRNA complex are disclosed, wherein the peptide based complex effectively enhances delivery of siRNA molecules into the cells and release of siRNA in the cell, and improves siRNA mediated gene silencing efficiency of cellular targets. Pharmaceutical compositions that include the complex, as well as a use of the complex in the gene therapy field, are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCESTATEMENT

This application is a continuation of U.S. Ser. No. 15/106,108, filedJun. 17, 2016; which is a US national stage application filed under 35USC § 371 of International Application No. PCT/CN2014/094198, filed Dec.18, 2014; which claims priority to Chinese Application No. CN20130698871.0, filed Dec. 18, 2013. The entire contents of each of theabove-referenced applications are hereby expressly incorporated hereinby reference.

BACKGROUND

Over the past decade, we have witnessed tremendous progress in ourunderstanding of the role of RNA molecules in the regulation of geneexpression. The main contribution to this progress was offered by thediscovery of RNA interference (RNAI). First identified in C. elegans byFire and Mello, RNAi is an evolutionary conserved mechanism that bringsabout a sequence specific, post transcriptional gene silencing (PTGS)through the use of short RNAs. RNA interference (RNAi) is a shortinterfering RNA (siRNA) mediated gene siliencing technology withspecific sequence. siRNA is duplex small molecule consisting of of 21-23nucleotides. Because RNAi has efficient and specific effect on genesilencing to disease, it has been gradually developed to a new genetreatment method for inherited or acquired diseases which includes viralinfections and cancer, and so on. Till now, a lot of animal treatmentexperiments and clinical trials have been carried out on using siRNA totreat disease.

The major limitations for the use of siRNA both in vitro and in vivo arethe instability of naked siRNA in physiological conditions, rapidclearance from the bloodstream, and the inability to cross the cellularmembrane to gain access to the intracellular environment. Although siRNAhas shown good silencing effect on in vitro application research and thecell level, because of its large molecular weight and a large number ofnegative charges carried by itself, it is not able to penetrate throughthe cell membrane and enter into the cell, non-unique target losingeffects and immune response will be initiated in the transportation. Inthe meanwhile, it also faces some other obstacles, such as nucleasedegradation and so on, which results tremendous challenges in diseasestreatment for siRNA transport system. So the transfection of siRNA hasbecome the main bottleneck and restricted its application. How toenhance the ability of siRNA to penetrate the cell membrane and improveits stability and targeting in vivo are the urgent problems to be solvedin siRNA drug carrier. So safe and effective siRNA drug carrier designand synthetise has become an important direction on siRNA drug researchand development. Chemical modifications in the sugars, nucleobases, andthe phosphate ester backbone of siRNA have been applied to improve itsnuclease resistance without interfering with the silencing efficiency.Conjugation with hydrophobic functional groups has also enhanced thecellular uptake.

In comparison with chemical modifications of NAs, carrier-mediatedstrategies are emerging as a simple and fast means to formulate NAtherapeutics and protect them from degradation. The carriers, includingviral vectors and non-viral vectors, co-assembled or covalentlyconjugated with siRNA, The carriers are designed to enhance celltargeting, prolong drug circulation time, and improve membranepermeation. Because of the potential safety problems and high cost, widerange of clinical applications o viral vectors are greatly limited.Common delivery carrier of non-viral gene drug is positively chargedcationic compound, it includes polycation, positively chargedphospholipids, chitosan, albumin, dendritic macromolecules and peptide,etc., they compress the gene to assembled particles by the positivelycharged groups and the negatively charged phosphate groups on DNA/RNA,and make gene smoothly pass through all kinds of obstacles to completethe gene transfer, such as immune system escape, cell membranepenetration, endosome release, and so on. Although there are somearguments on what is a clear relationship between the construction andproperties and transfection efficiency of DNA/RNA complexes there arestill a lot of conditions determining the biocompatibility and theefficiency of the transfer DNA/RNA of the complexes according toexperience. RNA/DNA carrier system formed by simple mixture and onlyelectrostatic interactions is not stable, so a more stable and suitablesized carrier system is prerequisite to be designed for gene drugs tohave good effect the of gene drugs.

As a kind of biological molecules, peptides have a lot of advantages tobe a gene carrier that synthetic polymers does not have. Peptides have20 kinds of amino acids that have different characteristics to form aprimary sequence with tremendous properties; Beta folding, alpha helixand other secondary structure is obtainable by sequence design.Molecular with high purity, simple distribution and clear structure isobtainable by solid-phase synthesis. It is easy to be modified orconnected to cellular target sequence, and enhance specificy. Peptideshave been employed to deliver synthetic drugs, small molecules,bioactive peptides, therapeutic proteins, and NAs by a mechanism thathas not yet been fully understood. These peptides may includeprotein-derived cell penetrating peptides (CPPs), cationic peptides,designed amphiphilic peptides, fusogenic peptides, cell targetingpeptides (CTP) and peptides containing a nuclear localization signal.Cationic peptides rich in basic amino acids can electrostaticallyinteract with small NAs or condense NA into small stable particles. CPPscan facilitate the translocation of the complex through the cellmembrane. Histidine-rich pH-sensitive or fusogenic peptides can enhancethe endosomal escape and cytoplasmic release of the gene complex.Involvement of CTPs in gene delivery systems mediates cell and/ortissue-specific targeting. Finally, attachment of a NLS peptide improvesnuclear localization of the gene complex.

Although researches on siRNA drug delivery carrier has made somebreakthroughs and developments, there are still some key issues need tobe resolved to make siRNA drugs successfully been used in treatment ofhuman diseases. For example, toxicity, specificity, targeting, immunestimulation, low transfection efficiency, and so on. Thus, becausebiodegradable and bicharacteristic peptide carrier can effectivelycombine and protect siRNA molecules to enhance cellular uptake of siRNAmolecules, and improve its stability and targeting, and effectivelyrelease siRNA molecules, and reach the purpose of treatment, it makessuch carrier has better advantage and application prospect in siRNA drugdelivery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the silencing efficiency of GL5/siRNA complexes withmolar ratio 20/1 and 40/1 in CHO cells with siRNA concentrations of 50nM.

FIG. 2 illustrates the silencing efficiency of STR-KV/siRNA complexeswith molar ratio 30/1, 40/1, and 60/1 in CHO cells with siRNAconcentrations of 50 nM.

FIG. 3 illustrates the silencing efficiency of STR-HR/siRNA complexeswith molar ratio 30/1, 45/1, and 60/1 in CHO cells with siRNAconcentrations of 100 nM.

FIG. 4 illustrates the silencing efficiency of STR-C6M1/siRNA andSTR-C6M3/siRNA complexes with molar ratio 40/1, and 60/1 in CHO cellswith siRNA concentrations of 50 nM.

FIG. 5 illustrates cytotoxicity of GL5/siRNA complexes with molar ratio20/1 and 40/1 in CHO cells with siRNA concentrations of 50 nM.

FIG. 6 illustrates cytotoxicity of STR-KV/siRNA complexes with molarratio 30/1, 45/1, and 60/1 in CHO cells with siRNA concentrations of 50nM.

FIG. 7 illustrates cytotoxicity of STR-HR/siRNA complexes with molarratio 30/1, 45/1, and 60/1 in CHO cells with siRNA concentrations of 100nM.

FIG. 8 illustrates cytotoxicity of STR-C6M1/siRNA and STR-C6M3/siRNAcomplexes with molar ratio 40/1 and 60/1 in CHO cells, with siRNAconcentrations of 50 nM.

DETAILED DESCRIPTION

The presently disclosed and/or claimed inventive concept(s) is toprovide a peptide, the peptide can be used to form a complex with siRNAfor gene delivery.

In order to solve the above technical problem, the presently disclosedand/or claimed inventive concept(s) provides: a peptide comprisinghydrophobic part and hydrophilic part, wherein the hydrophilic partcomprises a positively charged amino acid residues.

In certain non-limiting embodiments, the total number of amino acidresidues contained in the peptide is 8-50.

In certain non-limiting embodiments, the peptide has the formula:GLWHxAWLWHyAFLASHzRLLRLLR; wherein 1≤x≤5, 1≤y≤5, 1≤z≤6; and wherein x,y, and z are integers (SEQ ID NO:10).

In certain non-limiting embodiments, the nitrogen terminal of thepeptide is acetylated, and carbon terminal of the peptide is amidated.

In certain non-limiting embodiments, the peptide isCH₃(CH₂)₁₆CONH—B—CONH₂, wherein B is an amino acid sequence.

In certain non-limiting embodiments, B is GLWHxAWLWHyAFLASHzRLLRLLR,1≤x≤5, 1≤y≤5, 1≤z≤6, x, y, z are integers (SEQ ID NO:10).

In certain non-limiting embodiments, B is HaKbVc, 3≤a≤8, 3≤b≤8, 6≤c≤8,a, b, c are integers (SEQ ID NO:11).

In certain non-limiting embodiments, B is HdRe, 8≤d≤30, 8≤e≤20, d, e areintegers (SEQ ID NO:12).

In certain non-limiting embodiments, B is RLWRLLWRLWRRLWRLLR (SEQ IDNO:4).

In certain non-limiting embodiments, B is RLWHLLWRLWRRLHRLLR (SEQ IDNO:5).

The presently disclosed and/or claimed inventive concept(s) alsoprovides a complex comprising a cargo molecule and the peptide of anyone above mentioned.

In certain non-limiting embodiments, the cargo molecule and the peptideare joined by non-covalent bonds.

In certain non-limiting embodiments, the particle size of the complex isranged from 20 nm to 999 nm.

In certain non-limiting embodiments, the cargo molecule is a nucleicacid.

In certain non-limiting embodiments, the nucleic acid is a shortinterfering RNA (siRNA).

In certain non-limiting embodiments, the siRNA is complexed with thepeptide at a molar ratio within the range of 1:1 to 80:1.

In certain non-limiting embodiments, the siRNA is complexed with thepeptide at a molar ratio of 20:1.

In certain non-limiting embodiments, the siRNA is complexed with thepeptide at a molar ratio of 40:1.

In certain non-limiting embodiments, the siRNA is complexed with thepeptide at a molar ratio of 60:1.

The presently disclosed and/or claimed inventive concept(s) alsoprovides a pharmaceutical composition comprising any one complexmentioned as above.

The presently disclosed and/or claimed inventive concept(s) alsoprovides a kit used to deliver drugs to patient, the kit comprising apharmaceutical composition as mentioned above, one or more electrolytes,buffers, a delivery device and a container adapted to mix one or moreother agents together.

The presently disclosed and/or claimed inventive concept(s) alsoprovides a use of the peptide mentioned as above to deliver siRNA intocells or tissues.

In certain non-limiting embodiments, the cell is ovary cell.

In certain non-limiting embodiments, the cell is a tumber cell or thetissue is tumber tissue.

The effect of the presently disclosed and/or claimed inventiveconcept(s) is, the peptide provided here is non-covalently coupled withsiRNA to conveniently generate a peptide/siRNA complex in the form ofnanoparticles. Certain peptides designed by the presently disclosedand/or claimed inventive concept(s) have both hydrophilic andhydrophobic properties, by the interaction of the hydrophilic andhydrophobic parts of the cell membrane bilayer, the delivery of siRNAinto the cells and the release of siRNA in the cell was effectivelyenhanced, and the siRNA mediated silencing efficiency of cellulartargets was improved.

The technical proposal and the advantages of the presently disclosedand/or claimed inventive concept(s) will be further described by thedetailed embodiments. But in any way it is not contributed to limit thescope of the presently disclosed and/or claimed inventive concept(s).

The presently disclosed and/or claimed inventive concept(s) provides apeptide comprising a hydrophobic part and a hydrophilic part. Thehydrophilic part comprises a positively charged amino acid residue. Thehydrophobic part includes hydrophobic amino acid residues, stearic acidor stearic acid and hydrophobic amino acid residues. The hydrophobicamino acid residues are selected from one or more glycine (G), alanine(A), leucine (L), isoleucine (I), aromatic tryptophan (W), phenylalanine(F) and valine (V) and its derivatives. The hydrophilic part is selectedfrom one or more arginine (R), histidine (H), lysine (k), serine (s) andits derivatives.

The peptide of the presently disclosed and/or claimed inventiveconcept(s) comprises peptide and peptide derivatives. Peptidederivatives comprise a peptide modified on nitrogen terminal or carbonterminal. Certain designed amphiphilic peptides possess both hydrophilicand hydrophobic moieties, therefore the peptide is bicharacteristic.Because of the peptide of the presently disclosed and/or claimedinventive concept(s) have unique sequences or groups, it is easy togenerate a secondary structure by self-assembly or co-assembly.

The presently disclosed and/or claimed inventive concept(s) alsoprovides a complex with the peptide mentioned as above and a cargomolecular. In certain non-limiting embodiments, the cargo molecule is anucleic acid. In particular non-limiting embodiments, the nucleic acidis siRNA.

The peptide of the presently disclosed and/or claimed inventiveconcept(s) is to be formed into nanoparticles by co-assembly with thenegatively charged siRNA. In certain non-limiting embodiments, thenanoparticles are selected from nano fibers, nano wires, nano films andnano spheres. In certain non-limiting embodiments, particle size of thecomplex formed by the peptide and siRNA is ranged from 20 nm to 999 nm.This is because only some certain sized nanoparticles are passablethrough the cell membrane. In certain non-limiting embodiments, thetotal number of amino acid residues contained in the peptide is rangedfrom 8 to 50. Peptide formed with amino acid within this range issuitable for forming nano structure after being assembled with siRNA.the peptide formed with this range of amino acid and siRNA assembled.

In one embodiment, the hydrophobic part is directly connected with thehydrophilic part. And they could also be joined via a connecting chainsegment. In certain non-limiting embodiments, the connecting chainsegment is one of glycine (G) and H (histidine).

In one embodiment, the peptide has a formula: GLWHxAWLWHyAFLASHzRLLRLLR,simply be regarded as GL5-M, 1≤x≤5, 1≤y≤5, 1≤z≤6, x, y, z are integers(SEQ ID NO:10). In certain non-limiting embodiments, nitrogen terminalof the GL5-M is acetylated, and carbon terminal of the GL5-M isamidated.

In one embodiment, the peptide is GLWHAWLWHAFLASHRLLRLLR (SEQ ID NO:1).In one embodiment, the peptide is CH₃CONH-GLWHAWLWHAFLASHR LLRLLR-CONH₂,simply be regarded as GL5. The GL5 is designed based on peptide GL1 thatis originally reported in international patent application publicationno. WO2013/075244 A1. GL1(GLWRAWLWKAFLASNWRRLLRLLR; SEQ ID NO:13)consists of a hydrophobic domain GLWRAWLWKAFLASNW (SEQ ID NO:14) and acationic domain RRLLRLLR (SEQ ID NO:15). The amphiphilic property ofthis peptide makes it able to self-assemble when dissolved in water andco-assemble with negatively charged siRNA to form functionalizednanoparticles. This peptide can achieve about 95% siRNA delivery(uptake) efficiency but its gene silencing efficiency is about 50%. Itis indicated that GL1 has some problems on siRNA release efficiency.Histidine (H) was reported to increase endosomal escape. Histidine wastaken to replace four amino acids of GL1 to form a new peptide sequenceGL5. The sequence of GL5 was further extended to a series of peptides bychanging the number of H. This new series of peptides were named asGL5-M.

In certain non-limiting embodiments, a nitrogen terminal of the peptideprovided by the presently disclosed and/or claimed inventive concept(s)is stearylated (simply named as STR), a carbon terminal of the peptideis amidated. STR is formed by dehydration of stearic acid and nitrogenterminal of amino. The stearic acid has been proven to have highaffinity with the cell membrane, and stearylation of the peptide is ableto increase the transfection efficiency of siRNA. In certainnon-limiting embodiments, peptide includes histidine and itsderivatives.

In certain non-limiting embodiments, peptide of the presently disclosedand/or claimed inventive concept(s) has a formula:CH₃(CH₂)₁₆CONH—B—CONH₂, B is an amino acid sequence, CH₃(CH₂)₁₆CONH isstearyl, and CONH₂ is amide. The stearyl is connected with the nitrogenterminal of the B, and the amide is connected with the carbon terminalof B.

In certain non-limiting embodiments, B has the formula:GLWHxAWLWHyAFLASHzRLLRLLR, 1≤x≤5, 1≤y≤5, 1≤z≤6, x, y, z are integers(SEQ ID NO:10). The formula of the peptide is:CH₃(CH₂)₁₆CONH-GLWHxAWLWHyAFLASHzRLLRLLR-CONH₂, named as STR-GL5-M. Theintroduction of stearic acid can effectively improve membranepenetration ability of the peptide.

In certain non-limiting embodiments, B has a formula: HaKbVc, 3≤a≤8,3≤b≤8, 6≤c≤8, a, b, c are integers (SEQ ID NO:11). Peptide has aformula: CH₃(CH₂)₁₆CONH—HaKbVc-CONH₂, named as STR-KV-M. In oneembodiment, B is HHHKKKVVVVVV (SEQ ID NO:2), peptide isCH₃(CH₂)₁₆CONH—HHHKKKVVVVVV—CONH₂, named as STR-KV. STR-KV peptideadopted a stearic acid as one of the hydrophobic moiety of STR-KV.Stearic acid has been proven to have high affinity with the cellmembrane, and the stearylation of cell penetrating peptides has beenreported to be able to increase the transfection efficiency for bothplasmid DNA and siRNA. The amino acid histidine is incorporated as alinker, i.e., HHH. Histidine is a pH sensitive amino acid and can beprotonated at low pH. Through the introduction of histidine moieties,peptide will act like a proton sponge that is able to disrupt endosomalmembranes, resulting in the release of siRNA complexes into cytosol. Thehigh positive charge density has been reported to result in toxicity,due to the fact that positively charged amino acid residues mightinteract with negatively charged cell membranes and cause aggregationand rupture on the membrane surface. Thus, three lysines are adopted inthe sequences, in order to co-assemble with siRNA molecules throughelectrostatic interactions. Polyelectrolyte complex is formed bycombining positively charged part with negative charge from phosphategroup of siRNA molecule through electrostatic interactions. The complexmakes the siRNA transport into the cell by stimulating non-specific cellendocytosis, and then dissociates siRNA molecules from transport carrierthrough “proton sponge” effect mediated endosome escape, so as torealize the transportation of siRNA. Another hydrophobic part is addedto the sequence, consisting of six valine residues. The structure withtwo hydrophobic tails has been employed in the lipid-based genedelivery, e.g., DOPE, DOPC. Lipid tails are thought to be important foreffective gene delivery, and can assist in cellular uptake throughinteracting with the hydrophobic tails in the lipid bilayer of thecells. The lipids consisting of two hydrophobic tails have been reportedto have lower critical aggregation concentrations than single-chainlipids. The hydrophobic block with six valine residues is adopted tomimic the structure of two lipid tails. It is beneficial to penetratethe cell membrane.

In certain non-limiting embodiments, B has a formula: HdRe, 8≤d≤30,8≤e≤20, d, e are integers (SEQ ID NO:12). Peptide has a formula:CH₃(CH₂)₁₆CONH—HdRe—CONH₂, named as STR-HR-M. In one embodiment, B isHHHHHHHHHHHHHHHHRRRRRRRR (SEQ ID NO:3), peptide isCH₃(CH₂)₁₆CONH—HHHHHHHHHHHHHHHHRRRRRRRR—CONH₂, named as STR-HR. TheSTR-HR peptide is a novel molecular sequence designed by high throughputscreening. STR-HR consists of three parts: hydrophobic stearic acid, ahydrophobic tail, a segment of sixteen histidines (H16); and eightarginines (R8), a hydrophilic segment. The sequence R8 is a cellpenetrating peptide, which can strongly interact with cell membranes.Stearylation and the use of oligohistidines may facilitate the endosomalescape of peptide/siRNA complexes.

In one embodiment, B is RLWRLLWRLWRRLWRLLR (SEQ ID NO:4), the peptide isCH₃(CH₂)₁₆CONH—RLWRLLWRLWRRLWRLLR-CONH₂, named as STR-C6M1.

In one embodiment, B is RLWHLLWRLWRRLHRLLR (SEQ ID NO:5), the peptide isCH₃(CH₂)₁₆CONH—RLWHLLWRLWRRLHRLLR-CONH₂, named as STR-C6M1.

STR-C6M1 and STR-C6M3 are derivatives from the peptides C6M1 and C6M3,respectively. C6M1 is RLWRLLWRLWRRLWRLLR (SEQ ID NO:4), C6M3 isRLWHLLWRLWRRLHRLLR (SEQ ID NO:5). C6M1 and C6M3 were originally reportedin international patent application publication no. WO2013/075244 A1.Arginine rich peptides are reported to be capable of delivering siRNAsinto cells with high efficiency and low toxicity, while at least fivearginine residues are required to maintain the transfection efficiency.Hydrophobic residues such as leucine are also included in C6 sequence.These hydrophobic amino acids can facilitate the translocation ofpeptide by interacting with the hydrophobic tails in the lipid bilayeror assisting in pore formation in the cell membrane. It has been foundthat alanine, leucine and histidine are found abundantly in the helicalregions of proteins. They indicate that leucine is the strongeststructure forming residue in the proteins investigated. To increase itssolubility and transfection efficiency, we substituted some amino acidresidues with aromatic tryptophan and histidine. Stearic acid is one ofthe fatty acids occurring abundantly in the body and has highinteraction ability with the cell membrane. Modified C6M3 and C6M1 andstearic acid further enhance penetration to the cell membrane.

The presently disclosed and/or claimed inventive concept(s) alsoprovides a pharmaceutical composition. The pharmaceutical compositioncomprises the complexes of peptide and cargo molecule. In certainnon-limiting embodiments, the cargo molecule is selected from one ormore siRNA, miRNA and shRNA. In particular non-limiting embodiments, thecargo molecule is siRNA. In one embodiment, the pharmaceuticalcomposition of the presently disclosed and/or claimed inventiveconcept(s) can reduce the level of endogenous protein in cell or tissue.In certain non-limiting embodiments, the cell is a tumber cell or thetissue is tumber tissue.

The presently disclosed and/or claimed inventive concept(s) alsoprovides a kit used to deliver drugs for patient, the kit comprises apharmaceutical composition, one or more electrolytes, buffers, adelivery device and a container that mixes one or more other agentstogether. The kit also includes instructions for using of thepharmaceutical, a mixture of pharmaceutical and other reagents, and thedescription to be given to the user.

The presently disclosed and/or claimed inventive concept(s) alsoprovides a use of the peptide as a carrier to deliver siRNA to cells ortissues. In one embodiment, the cell is CHO. In one embodiment, the cellis a tumber cell. In one embodiment, the tissue is tumber tissue.

Experimental Apparatus and Sample Preparation

1. Experimental Apparatus

The molar concentration of siRNA was measured by the nucleic acidprotein analyzer Nanodrop ND-1000, purchased from the ThermoFishercompany. The number of mRNA was measured by Mx3005P Real-Time PCR SystemAgilent Co (Wilmington, USA).

2. Sample

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are used as the targetgene. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a housekeepinggene widely expressed in diverse tissues and cell types and functions ina variety of cellular processes. The corresponding siRNA, i.e.,Silencer® GAPDH siRNA (Human, Mouse, Rat), was purchased from Ambion.All peptide compounds was purchased from Pepscan Systems (Leystad,Netherlands).

3. Cell Test

An adult CHO tissue CHO-K1 (ATCC CCL-61) has been used as a cell testobject.

4. Detection of mRNA

Real-time RT-PCR is at present the most sensitive method for thedetection of low abundance mRNA. Total RNA from the cells was extractedwith TRIzol reagent, then treated with chloroform and 2-propanol asrecommended by the manufacturer. The RNA concentrations were measured byNanodrop. All the RNAs were reverse transcribed with Bio-Rad iScriptcDNA synthesis kit according to the protocol. The cDNA synthesis wasprimed with a unique blend of oligo (dT) and random primers. Thesequences of the primers used for the mouse GAPDH gene are5′-TTGCTGTTGAAGTCGCAGGAG-3′ (SEQ ID NO:6) and 5′-TGTGTCCGTCGTGGATCTGA-3′(SEQ ID NO:7)(Sigma, Oakville, Ontario, Canada). Cyclophilin, ahouse-keeping gene, was used as an internal control to normalize theGAPDH gene expression. Mouse cyclophilin mRNA amplified using thefollowing primers: 5′-AGGGTTTCTCCACTTCGATCTTGC-3′ (SEQ ID NO:8) and5′-AGATGGCACAGGAGGAAAGAGCAT-3′ (SEQ ID NO:9)(Sigma, Oakville, Ontario,CA).

5. Cytotoxicity Test

CHO-K1 cells were seeded at 5,000-8,000 cells/well in clear,flat-bottomed, 96-well plates in triplicate and grown for 24 h at 37° C.with 5% CO₂. The complexes or control samples were added to the cells in150 μl of standard media and the cells were incubated for 48 h. The CCK8assay was used to determine cell viability, according to themanufacturer's instructions. Briefly, 110 μl of CCK8 stock was added toeach well and incubated for 2 h at 37° C. in 5% CO₂. Then, the cultureswere removed from the incubator and the absorbance at 570 nm was read ona plate reader (FLUOstar OPTIMA, BMG, Germany). The backgroundabsorbance of the multiwell plates at 690 nm was determined andsubtracted from the 570 nm measurement. The results obtained fromtriplicate wells were averaged and normalized using the value obtainedfor non-treated cells.

All the professional and scientific terms used in the presentlydisclosed and/or claimed inventive concept(s) are the same as that ofthe person skilled in the field. In addition, any method or materialwhich is similar or equivalent to the written contents can be applied tothe method of the presently disclosed and/or claimed inventiveconcept(s). The particular (but non-limiting) embodiments illustratedhere are only for examples.

Combining with the following example, a more detailed description of thecontent of the presently disclosed and/or claimed inventive concept(s)is provided. It should be understood that the implementation of thepresently disclosed and/or claimed inventive concept(s) is not limitedto the detailed embodiments as below, and any amendments and/or changesunder the spirit of the presently disclosed and/or claimed inventiveconcept(s) shall fall into the protection scope of the presentlydisclosed and/or claimed inventive concept(s). In this presentlydisclosed and/or claimed inventive concept(s), unless specified, allpercentages in the description are directed to weight ration, allequipments and materials can be purchased from the market or commonlyused in the industry.

Example 1-1

GL5 peptide is used as the carrier of siRNA delivery in this embodiment.

Preparation of siRNA/GL5 GAPDH Complex

GL5 peptide with concentration of 1000 nM and GAPDH siRNA withconcentration of 50 nM were dissolved respectively in a serum freeOpti-MEM solution with the molar ratio of 20:1. Then the gene was addedinto the peptide solution by the method of uniform mixing. Afterstanding half an hour, the carrier is ready in a fully stable status tobe measured and for further use.

In Vivo Transfection of siRNA/GL5 GAPDH Complex

The cells were seeded with a confluency of 35,000-40,000 cells/well inF12K medium with 10% FBS without antibacterial agents, 24 hrs beforetransfection. On the next day, rinse the cells with PBS buffer beforetransfection and add 200 μl of Opti-MEM. 100 μl of the complex solution(GAPDH siRNA-peptide) were added to each well. The cells were incubatedwith the complex at 37° C. in an incubator. After 4 hours, 300 μl F12-Kwith 20% FBS was added without removing the transfection mixture. Thecells were rinsed and lysed 48 hours after the start of transfection.

Detect mRNA content in cells by Real-time RT-PCR, and proceed thecytotoxicity test.

Example 1-2

GL5 peptide with concentration of 2000 nM and GAPDH siRNA withconcentration of 50 nM are used to prepare of GL5/siRNA complex with themolar ratio of 40:1, the preparation method is same as that of example1-1.

In vivo transfection of GL5/siRNA complex, the method is same as that ofexample 1-1.

Detect mRNA content in cells by Real-time RT-PCR, and proceed thecytotoxicity test.

Example 2-1

STR-KV peptide is used as the carrier of siRNA delivery in thisembodiment.

STR-KV peptide with concentration of 1500 nM and GAPDH siRNA withconcentration of 50 nM are used to prepare of STR-KV/siRNA complex withthe molar ratio of 30:1, the preparation method is same as that ofexample 1-1.

In vivo transfection of STR-KV/siRNA complex, the method is same as thatof example 1-1.

Detect mRNA content in cells by Real-time RT-PCR, and proceed thecytotoxicity test.

Example 2-2

STR-KV peptide is used as the carrier of siRNA delivery in thisembodiment.

STR-KV peptide with concentration of 2250 nM and GAPDH siRNA withconcentration of 50 nM are used to prepare of STR-KV/siRNA complex withthe molar ratio of 45:1, the preparation method is same as that ofexample 1-1.

In vivo transfection of STR-KV/siRNA complex, the method is same as thatof example 1-1.

Detect mRNA content in cells by Real-time RT-PCR, and proceed thecytotoxicity test.

Example 2-3

STR-KV peptide is used as the carrier of siRNA delivery in thisembodiment.

STR-KV peptide with concentration of 3000 nM and GAPDH siRNA withconcentration of 50 nM are used to prepare of STR-KV/siRNA complex withthe molar ratio of 60:1, the preparation method is same as that ofexample 1-1.

In vivo transfection of STR-KV/siRNA complex, the method is same as thatof example 1-1.

Detect mRNA content in cells by Real-time RT-PCR, and proceed thecytotoxicity test.

Example 3-1

STR-HR peptide is used as the carrier of siRNA delivery in thisembodiment.

STR-HR peptide with concentration of 3000 nM and GAPDH siRNA withconcentration of 100 nM are used to prepare of STR-KV/siRNA complex withthe molar ratio of 30:1, the preparation method is same as that ofexample 1-1.

In vivo transfection of STR-HR/siRNA complex, the method is same as thatof example 1-1.

Detect mRNA content in cells by Real-time RT-PCR, and proceed thecytotoxicity test.

Example 3-2

STR-HR peptide is used as the carrier of siRNA delivery in thisembodiment.

STR-HR peptide with concentration of 4500 nM and GAPDH siRNA withconcentration of 100 nM are used to prepare of STR-KV/siRNA complex withthe molar ratio of 45:1, the preparation method is same as that ofexample 1-1.

In vivo transfection of STR-HR/siRNA complex, the method is same as thatof example 1-1.

Detect mRNA content in cells by Real-time RT-PCR, and proceed thecytotoxicity test.

Example 3-3

STR-HR peptide is used as the carrier of siRNA delivery in thisembodiment.

STR-HR peptide with concentration of 6000 nM and GAPDH siRNA withconcentration of 100 nM are used to prepare of STR-KV/siRNA complex withthe molar ratio of 60:1, the preparation method is same as that ofexample 1-1.

In vivo transfection of STR-HR/siRNA complex, the method is same as thatof example 1-1.

Detect mRNA content in cells by Real-time RT-PCR, and proceed thecytotoxicity test.

Example 4-1

STR-C6M1 peptide is used as the carrier of siRNA delivery in thisembodiment.

STR-C6M1 peptide with concentration of 2000 nM and GAPDH siRNA withconcentration of 50 nM are used to prepare of STR-KV/siRNA complex withthe molar ratio of 40:1, the preparation method is same as that ofexample 1-1.

In vivo transfection of STR-C6M1/siRNA complex, the method is same asthat of example 1-1.

Detect mRNA content in cells by Real-time RT-PCR, and proceed thecytotoxicity test.

Example 4-2

STR-C6M1 peptide is used as the carrier of siRNA delivery in thisembodiment.

STR-C6M1 peptide with concentration of 3000 nM and GAPDH siRNA withconcentration of 50 nM are used to prepare of STR-KV/siRNA complex withthe molar ratio of 60:1, the preparation method is same as that ofexample 1-1.

In vivo transfection of STR-C6M1/siRNA complex, the method is same asthat of example 1-1.

Detect mRNA content in cells by Real-time RT-PCR, and proceed thecytotoxicity test.

Example 5-1

STR-C6M3 peptide is used as the carrier of siRNA delivery in thisembodiment.

STR-C6M3 peptide with concentration of 2000 nM and GAPDH siRNA withconcentration of 50 nM are used to prepare of STR-KV/siRNA complex withthe molar ratio of 40:1, the preparation method is same as that ofexample 1-1.

In vivo transfection of STR-C6M3/siRNA complex, the method is same asthat of example 1-1.

Detect mRNA content in cells by Real-time RT-PCR, and proceed thecytotoxicity test.

Example 5-2

STR-C6M3 peptide is used as the carrier of siRNA delivery in thisembodiment.

STR-C6M3 peptide with concentration of 3000 nM and GAPDH siRNA withconcentration of 50 nM are used to prepare of STR-KV/siRNA complex withthe molar ratio of 60:1, the preparation method is same as that ofexample 1-1.

In vivo transfection of STR-C6M3/siRNA complex, the method is same asthat of example 1-1.

Detect mRNA content in cells by Real-time RT-PCR, and proceed thecytotoxicity test

Contrast Example 1

Lipid (lipofactamine 2000, lipo) is used as the carrier of siRNAdelivery in this embodiment.

Prepare lipid/siRNA complex, the preparation method is same as that ofexample 1-1.

In vivo transfection of Lipid/siRNA GAPDH complex, the method is same asthat of example 1-1.

Detect mRNA content in cells by Real-time RT-PCR, and proceed thecytotoxicity test

RT-PCR results of GAPDH siRNA complexed with different peptides atdifferent molar ratio are shown in FIGS. 1 to 4. FIG. 1 shows theintracellular silencing of GAPDH gene in CHO cells where siRNA werecomplexed with GL5 at a molar ratio of 20/1 and 40/1. FIG. 2 shows theintracellular silencing of GAPDH gene in CHO cells where siRNA werecomplexed with STR-KV at a molar ratio of 30/1, 40/1 and 60/1. FIG. 3shows the silencing efficiency of STR-HR/siRNA complexes with molarratio 30/1, 45/1, and 60/1 in CHO cells. FIG. 4 shows the silencingefficiency of STR-C6M1/siRNA and STR-C6M3/siRNA complexes with molarratio 40/1, and 60/1 in CHO cells. The results shown in FIGS. 1 to 4correspond to an average of at least three separate experiments.

Cytotoxicity results of GAPDH siRNA complexed with different peptides atmolar ratio 20 to 60 are shown in FIGS. 5 to 8. FIG. 5 shows thecytotoxicity in CHO cells where siRNA were complexed with GL5 at a molarratio of 20/1 and 40/1. FIG. 6 shows the intracellular silencing ofGAPDH gene in CHO cells where siRNA were complexed with STR-KV at amolar ratio of 30:1, 45:1 and 60:1. FIG. 7 shows the silencingefficiency of STR-HR/siRNA complexes with molar ratio 30/1, 45/1, and60/1 in CHO cells. FIG. 8 shows the cytotoxicity in CHO cells wheresiRNA were complexed with STR-C6M1 and STR-C6M3 at a molar ratio of 40/1and 60/1. The results shown in FIGS. 5 to 8 correspond to an average ofat least three separate experiments.

With reference to FIG. 1 and FIG. 5, when the molar ratio of GL5 andsiRNA is 20/1 and 40/1, the gene silencing efficiency of siRNA is 41%and 65%, respectively, which is lower than that of the lipid transfectedsiRNA. But for both GL5 itself and the GL5/siRNA complex, the cellsurvival rates are about 105% and 104% respectively, which are muchhigher than that of the lipid or lipid/siRNA complex, which is 58%. GL5peptide of the presently disclosed and/or claimed inventive concept(s)is modified based on GL1 peptide in the prior art. Four positivelycharged histidine are taken to replace four amino acids on the basis ofGL1 to get GL5 peptide. And histidine has been proven to have highendosomal escape ability, it delivers the siRNA entered into the cell torelease from endosomal and into cytoplasm, thereby results silencingeffect with mRNA. According to disclosure of international patentapplication Publication No. WO2013/075244A1, gene silencing efficiencyof siRNA with GL1 carrier is about 50%. Therefore, comparing with theprior art, silencing efficiency and biological compatibility of thepresently disclosed and/or claimed inventive concept(s) have got greatenhancement.

Shown as FIG. 2 and FIG. 6, the gene silencing efficiencies areincreased with molar ratio of STR-KV/siRNA increase from 30/1 to 45/1and 60/1, which are 47%, 65%, 73%, respectively. They are close to thegene silencing efficiency of lipid/siRNA complex under the samecondition, which is 85%. Similarly, the cell survival rates of STR-KVand STR-KV/siRNA are approximately 98%, 97% and 90%, respectively, whichwas slightly decreased with the increase of molar ratio, but were higherthan that of the lipid or lipid/siRNA complex, which is 75%. In summary,the peptide STR-KV designed by the presently disclosed and/or claimedinventive concept(s) has good siRNA delivery performance and excellentbiocompatibility, and it is suitable for application in vivo.

Shown as FIG. 3 and FIG. 7, the gene silencing efficiencies ofSTR-HR/siRNA are increased with the molar ratio increase from 30/1, to45/1 and 60/1, which are 50%, 67%, 87% respectively. They are higherthan that of the lipid transfected siRNA at the molar ratio of 60/1,which is 85%. The cell survival rate of STR-HR and STR-HR/siRNA complexreaches the maximum when the molar ratio is 45/1, but the cell survivalrates in the three molar ratio are higher than that of the lipid orlipid/siRNA complex. At the same time, it was found that the absorptionefficiency of STR-HR was higher than that of lipid 2000 according toresult of cell absorption test. Silencing efficiency of STR-HR/siRNAcomplex is similar to that of lipid 2000/siRNA complex. Cytotoxicity ofSTR-HR per molar ratio for silencing efficiency test was not higher thanthat of lipid 2000. In addition, by dynamic light scatteringexperiments, it is proven that particles size of STR-HR and siRNAcomplexes are all in a range from 50 nm to 150 nm when molar ratio rangeof STR-HR/siRNA is within 15-60. Such particle size range is suitablefor applications in vivo through intravenous control.

Molar ratio range of STR-HR/siRNA is from 15 to 60, Zeta potential isfrom 21 mv to 28 mv companying with the molar ratio increase. Asdescribed above, the peptide STR-HR designed by the presently disclosedand/or claimed inventive concept(s) has excellent siRNA transferperformance and biocompatibility, wherein, STR-HR/siRNA with a molarratio of 60/1 has the best effect.

Shown as FIG. 4 and FIG. 8, the gene silencing efficiencies ofSTR-C6M1/siRNA are increased with the molar ratio increase from 40/1, to60/1, which are 47% and 49% respectively. They are higher than that ofthe lipid transfected siRNA. The cell survival rates of STR-C6M1 andSTR-C6M1/siRNA are approximately 93% and 88% respectively, which wasslightly decreased with the increase of molar ratio, but were muchhigher than that of lipid or lipid/siRNA complex. In addition, ininternational patent application WO2013075244, cell survival rate ofC6M3/siRNA transfected with molar ratio of 40/1 is less than 40%, whichwas far less than that of the presently disclosed and/or claimedinventive concept(s). Also, shown as FIG. 4 and FIG. 8, the genesilencing efficiencies of STR-C6M3/siRNA are increased with the molarratio increase from 40/1 to 60/1, which are 50%, 61% respectively. Theyare lower than that of the lipid transfected siRNA. The cell survivalrates of STR-C6M3 and STR-C6M3/siRNA are slightly decreased with themolar ratio increase, which are approximately 96% and 91%, respectively.But they are higher than that of the lipid or lipid/siRNA complex. Inaddition, in international patent application WO2013075244, cellsurvival rate of C6M3/siRNA transfected with molar ratio of 40/1 is lessthan 50%, which was far less than the cell survival rate of thepresently disclosed and/or claimed inventive concept(s). It isillustrated that after N terminal of the peptide is stearic acylated,the peptide STR-C6M1 and STR-C6M3 have good siRNA delivery performanceand excellent biocompatibility, and are suitable for application invivo.

In conclusion, the presently disclosed and/or claimed inventiveconcept(s) provides a peptide. It can be coupled non-covalently withsiRNA by self-assemble or co-assemble, and to be formed intonanostructures and to transfer siRNA into the cytoplasm. Because ofquickly protonated, biodegradable and bicharacteristic peptide orpeptide carrier can effectively combine and protect siRNA molecules toenhance cellular uptake of siRNA molecules, and improve its stabilityand targeting, and effectively release siRNA molecules, and reach thepurpose of treatment. Experiment result shows that, silence efficiencyof SiRNA is obviously improved, the peptide has the low toxicity andgood biocompatibility. The peptide carrier of the presently disclosedand/or claimed inventive concept(s) has better advantage and applicationprospect in siRNA drug delivery.

The above mentioned are only particular, non-limiting embodiments of thepresently disclosed and/or claimed inventive concept(s), they are notintended to be limited to the presently disclosed and/or claimedinventive concept(s), any modifications, equivalent replacements andimprovements within the spirit and principle of the presently disclosedand/or claimed inventive concept(s) shall be included in the protectionscope of the presently disclosed and/or claimed inventive concept(s).

What is claimed is:
 1. A peptide comprising a hydrophobic part and ahydrophilic part, wherein the hydrophilic part comprises a positivelycharged amino acid residue, wherein the peptide isCH₃(CH₂)₁₆CONH—B—CONH₂, and wherein B is SEQ ID NO:4 or
 5. 2. A complexcomprising: a cargo molecule; and a peptide comprising a hydrophobicpart and a hydrophilic part, wherein the hydrophilic part comprises apositively charged amino acid residue, wherein the peptide isCH₃(CH₂)₁₆CONH—B—CONH₂, and wherein B is SEQ ID NO:4 or
 5. 3. Thecomplex according to claim 2, wherein the cargo molecule and the peptideare coupled non-covalently.
 4. The complex according to claim 2, whereinthe particle size of the complex is in a range of from about 20 nm toabout 999 nm.
 5. The complex according to claim 2, wherein the cargomolecule is a nucleic acid.
 6. The complex according to claim 5, whereinthe nucleic acid is a short interfering RNA (siRNA).
 7. The complexaccording to claim 6, wherein the siRNA is complexed with the peptide ata molar ratio within a range of from about 1:1 to about 80:1.
 8. Thecomplex according to claim 6, wherein the siRNA is complexed with thepeptide at a molar ratio of about 20:1.
 9. The complex according toclaim 6, wherein the siRNA is complexed with the peptide at a molarratio of about 40:1.
 10. The complex according to claim 6, wherein thesiRNA is complexed with the peptide at a molar ratio of about 60:1. 11.A pharmaceutical composition containing a complex comprising: a cargomolecule; and a peptide comprising a hydrophobic part and a hydrophilicpart, wherein the hydrophilic part comprises a positively charged aminoacid residue, wherein the peptide is CH₃(CH₂)₁₆CONH—B—CONH₂, and whereinB is SEQ ID NO:4 or
 5. 12. A kit used to deliver drugs to a patient,wherein the kit comprises: a pharmaceutical composition comprising acomplex comprising: a cargo molecule; and a peptide comprising ahydrophobic part and a hydrophilic part, wherein the hydrophilic partcomprises a positively charged amino acid residue, wherein the peptideis CH₃(CH₂)₁₆CONH—B—CONH₂, and wherein B is SEQ ID NO:4 or 5; one ormore electrolytes; one or more buffers; a delivery device; and acontainer adapted to mix one or more other agents together.
 13. The kitaccording to claim 12, wherein the cargo molecule and the peptide arecoupled non-covalently.
 14. The kit according to claim 12, wherein theparticle size of the complex is in a range of from about 20 nm to about999 nm.
 15. The kit according to claim 12, wherein the cargo molecule isa nucleic acid.
 16. The kit according to claim 15, wherein the nucleicacid is a short interfering RNA (siRNA).
 17. The kit according to claim16, wherein the siRNA is complexed with the peptide at a molar ratiowithin a range of from about 1:1 to about 80:1.
 18. The kit according toclaim 16, wherein the siRNA is complexed with the peptide at a molarratio in a range of from about 20:1 to about 60:1.
 19. The kit accordingto claim 16, wherein the siRNA is complexed with the peptide at a molarratio of about 20:1 or about 40:1.
 20. The kit according to claim 16,wherein the siRNA is complexed with the peptide at a molar ratio ofabout 60:1.